1. Field of the Invention
The present invention is directed to methods and compositions for treating pathologies associated with hyperproliferative disorders. Among the hyperproliferative disorders which may be treated according to the invention are various tumors and cardiovascular diseases, such as vascular restenosis resulting from mechanical injury at an angioplasty site during treatment of an atheroscerotic lesion.
Atherosclerosis is a complex, polygenic disease which is defined in histological terms by deposits (lipid or fibrolipid plaques) of lipids and of other blood derivatives in blood vessel walls, especially the large arteries (aorta, coronary arteries, carotid). These plaques, which are more or less calcified according to the degree of progression of the atherosclerotic process, may be coupled with lesions and are associated with the accumulation in the vessels of fatty deposits consisting essentially of cholesterol esters. These plaques arc accompanied by a thickening of the vessel wall, hypertrophy of the smooth muscle, appearance of foam cells and accumulation of fibrous tissue. The atheromatous plaque protrudes markedly from the wall, endowing it with a stenosing character responsible for vascular occlusions by atheroma, thrombosis or embolism, which occur in those patients who are most affected. These lesions can lead to very serious cardiovascular pathologies such as infarction, sudden death, cardiac insufficiency, and stroke.
The technique of angioplasty has been developed to permit a non-surgical intervention of the atherosclerotic plaque. However, the treatment of an atherosclerotic lesion by angioplasty results very frequently (up to 50% of cases in some studies) in a restenosis following mechanical injury of the arterial wall A key event in this mechanism is the proliferation and migration of vascular smooth muscle cells (VSMC) from the media to the intima, as a result of the absence of protection and/or feedback control exercised by the endothelial cells of the intima.
Treatment of restenosis by administration of chemical or proteinaceous substances capable of killing vascular smooth muscle cells has been proposed. For example, psolaren derivatives, incorporated by proliferative cells and then sensitizing these cells to the action of light, have been used (March et al., 1993, Circulation, 87:184-191). Similarly, some cytotoxins consisting of a fusion protein between a plant or bacterial toxin fragment and a growth factor have also been used (Pickering et al., J. Clin. Invest., 1993, 91:724-729; Biro et al., 1992, Circ, Res., 71:640-645; Casscells et al., Proc. Natl. Acad. Sci. USA, 1992, 89:7159-7163). However, these treatments have many drawbacks, such as their low specificity, their indifferent efficacy; a considerable delay in acting and a potential toxicity. The present invention provides an effective, gene therapy approach for the treatment of hyperproliferative disorders, including restenosis.
2. Description of Related Art
Various genes have been isolated which are linked to the arrest of cell division. The gas (growth-arrest specific; gas 1-6) and gadd (growth-arrest and DNA damage-inducible; gadd34, gadd45 and gadd153) genes are strongly expressed in quiescent cells, that is cells which are blocked in the G0 phase of the cell cycle (Schneider et al., Cell 1988, 54; 787-793, Del Sal et al., Cell 1992, 12:3514-3521; Cowled et al., Exp.Cell.Res. 1994, 211:197-202; Brancolini and Schneider, J.Cell.Biol, 1994, 124:743-756; Zhan et al., Mol.Cell.Biol. 1993, 13:4242-4250; Jackman et al., Cancer Res. 54:5656-5662, 1994). In agreement with these findings on gene expression, microinjection of the gas-1 protein blocks the synthesis of DNA (Del Sal et al., Cell, 1992, 70:595-607). Conversely, the addition of growth factors such as PDGF (platelet-derived growth factor) or foetal calf serum decreases the expression of these genes in in-vitro models (Coccia et al. Mol.Cell.Biol. 1992, 12:3514-3521). This specificity of expression in relation to the state of cell proliferation also appears to have its counterpart in vivo. Thus, the gas-1 gene is strongly expressed in the rat uterus following ovariectomy (Ferrero and Cairo, Cell.Biol.Int. 1993, 17, 857-862). In this same animal model, treatment with oestrogeos results in a cell proliferation which is reflected, within the uterus, in an increase in the expression of the proto-oncogene c-myc and by a decrease in the expression of the gas-1 gene. Similarly, in a hepatic model of proliferation/regeneration, expression of the gas-6 gene is strongly reduced four hours after partial heptatectomy, i.e. in the period of transition from G0 to G1; this expression returns to normal, probably once division of the hepatocytes has been initiated (Ferrero et al. J.Cell.Physiol. 1994, 158:263-269).
Homeobox genes encode transcription factors which, at a cellular level, control growth, differentiation, and migration. The homeobox gene GAX (growth arrest-specific homeobox) is expressed in adult cardiovascular tissues and in muscular embryonic tissues. The GAX gene was initially identified in a cDNA library prepared from rat aorta. It encodes a protein of 303 amino acids. Its sequence has been characterized and its cDNA has been cloned (Gorski et al., Mol.Cell.Biol. 1993, 6, 3722-3733) The GAX homeobox gene is normally expressed in quiescent VSMCs and rapidly downregulated under conditions that induce VSMC dedifferentiation and proliferation. The GAX gene possesses certain properties which are similar to those of the gas and gadd genes, since it also appears to regulate the G0/G1 transition in the cell cycle. In the same way, the levels of GAX mRNA are decreased in the rat VSMCs by a factor of 10 after two hours of exposure to PDGF (Gorski et al., Mol.Cell.Biol. 1993, 6, 3722-3733). Expression of the GAX gene is therefore repressed during the VSMC mitogenic response. Gax expression is also rapidly down-regulated in vascular tissue immediately following balloon injury.
The present invention is directed to viral vectors comprising a GAX gene, compositions including the same, and using said composition for specifically arresting cell division. The GAX gene possesses properties which are particularly advantageous for use in the gene therapy of hyperproliferative disorders, in particular restenosis, by overexpression of the GAX gene in the vascular wall.
Methods of the invention comprise blocking proliferation of vascular smooth-muscle cells (VSMC) by in vivo delivery of a GAX gene in a viral vector, preferably replication defective recombinant adenoviral vectors.
The invention provides replication defective recombinant adenoviruses comprising at feast one inserted gene encoding all or part of a GAX protein or a variant thereof.
The invention provides methods for the treatment or prevention of a pathology linked to a hyperproliferative disorder, said method comprising administration of a replication defective recombinant adenovirus comprising at least one inserted gene encoding all or part of a GAX protein or a variant thereof.
The invention provides a method of treating restenosis comprising administering to a patient in need of such treatment a replication defective recombinant adenovirus comprising a GAX gene, in an amount effective to inhibit vascular smooth muscle cell proliferation and migration at a predetermined site. More preferably, the site is a site of mechanical injury to an arterial wall produced by treatment of an atherascterotic lesion by angioplasty.
The invention provides pharmaceutical compositions comprising one or more replication defective recombinant adenoviruses comprising at least one inserted gene encoding all or part of a GAX protein or a variant thereof.
These and other embodiments of the invention are discussed in detail below.